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Background: Preconditioning by intermittent fasting is linked to improved cognition and motor function, and enhanced recovery after stroke. Although the duration of fasting was shown to elicit different levels of neuroprotection after ischemic stroke, the impact of time of fasting with respect to the circadian cycles remains unexplored., Methods: Cohorts of mice were subjected to a daily 16-hour fast, either during the dark phase (active-phase intermittent fasting) or the light phase (inactive-phase intermittent fasting) or were fed ad libitum. Following a 6-week dietary regimen, mice were subjected to transient focal cerebral ischemia and underwent behavioral functional assessment. Brain samples were collected for RNA sequencing and histopathologic analyses., Results: Active-phase intermittent fasting cohort exhibited better poststroke motor and cognitive recovery as well as reduced infarction, in contrast to inactive-phase intermittent fasting cohort, when compared with ad libitum cohort. In addition, protection of dendritic spine density/morphology and increased expression of postsynaptic density protein-95 were observed in the active-phase intermittent fasting., Conclusions: These findings indicate that the time of daily fasting is an important factor in inducing ischemic tolerance by intermittent fasting., Competing Interests: None.

Background: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress., Methods: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively., Results: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia., Conclusions: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke., Competing Interests: Disclosures Dr Wainger receives research funding from argenx and is a consultant for QurAlis Corporation. The other authors report no conflicts.

Background: Ischemic stroke is often accompanied by oxidative stress and inflammatory response, both of which work synergistically to exacerbate the disruption of the blood-brain barrier and ischemic brain injury. ALK (anaplastic lymphoma kinase), a cancer-associated receptor tyrosine kinase, was found to play a role in oxidative stress and inflammation. In this study, we investigated the role of ALK inhibition in a murine model of ischemic stroke., Methods: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery in mice with a filament. The ALK inhibitor alectinib was administered following the stroke. ALOX15 (arachidonic acid 15-lipoxygenase) was overexpressed by adenovirus injection. The immunohistochemistry, Western blot, oxidative stress, inflammation, blood-brain barrier leakage, infarct volume, and functional outcomes were determined., Results: We found that the expression of ALK was markedly increased in the neurovascular unit after cerebral ischemia. Treatment with the ALK inhibitor alectinib reduced the accumulation of reactive oxygen species, lipid peroxidation, and oxidative DNA, increased the vascular levels of antioxidant enzymes, inactivated the vascular NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome pathway, and reduced vascular inflammation (ICAM-1 [intercellular adhesion molecule-1] and MCP-1 [monocyte chemoattractant protein-1]) after ischemia. Moreover, alectinib reduced the loss of cerebrovascular integrity and blood-brain barrier damage, consequently decreasing brain infarction and neurological deficits. Furthermore, alectinib reduced stroke-evoked ALOX15 expression, whereas virus-mediated overexpression of ALOX15 abolished alectinib-dependent inhibition of oxidative stress and vascular inflammation, blood-brain barrier protection, and neuroprotection, suggesting the protective effects of alectinib for stroke may involve ALOX15., Conclusions: Our findings demonstrated that alectinib protects from stroke by regulating ischemic signaling cascades and suggest that ALK may be a novel therapeutic target for ischemic stroke., Competing Interests: Disclosures None.

Background: Stroke is a leading cause of morbidity and mortality worldwide. Neuroinflammation plays a key role in acute brain injury of ischemic stroke. MicroRNA-210 (miR210) is the master hypoxamir and regulates microglial activation and inflammation in a variety of diseases. In this study, we uncovered the mechanism of miR210 in orchestrating ischemic stroke-induced neuroinflammation through repression of TET2 (ten-eleven translocation methylcytosine dioxygenase 2) in the adult mouse brain., Methods: Ischemic stroke was induced in adult WT (wild type) or miR210 KO (miR210 deficient) mice by transient intraluminal middle cerebral artery occlusion. Injection of TET2 silencing RNA or miR210 complementary locked nucleic acid oligonucleotides, or miR210 KO mice were used to validate miR210-TET2 axis and its role in ischemic brain injury. Furthermore, the effect of TET2 overexpression on miR210-stimulated proinflammatory cytokines was examined in BV2 microglia. Post assays included magnetic resonance imaging scan for brain infarct size; neurobehavioral tests, reverse transcription-quantitative polymerase chain reaction, and Western blot for miR210; and TET2 levels, flow cytometry, and ELISA for neuroinflammation in the brain after stroke or microglia in vitro., Results: miR210 injection significantly reduced TET2 protein abundance in the brain, while miR210 complementary locked nucleic acid oligonucleotides or miR210 KO preserved TET2 regardless of ischemic brain injury. TET2 knockdown reversed the protective effects of miR210 inhibition or miR210 KO on ischemic stroke-induced brain infarct size and neurobehavioral deficits. Moreover, flow cytometry and ELISA assays showed that TET2 knockdown also significantly dampened the anti-inflammatory effect of miR210 inhibition on microglial activation and IL (interleukin)-6 release after stroke. In addition, overexpression of TET2 in BV2 microglia counteracted miR210-induced increase in cytokines., Conclusions: miR210 inhibition reduced ischemic stroke-induced neuroinflammatory response via repression of TET2 in the adult mouse brain, suggesting that miR210 is a potential treatment target for acute brain injury after ischemic stroke.

Background: Treatment for ischemic stroke can be offered beyond conventional time limits for patients with favorable computed tomography perfusion (CTP), but this is not universally available. We sought a threshold for brain attenuation on nonenhanced computed tomography (NECT) to differentiate CTP-defined penumbra vs core, and correlated NECT features with CTP., Methods: We retrospectively assessed consecutive patients presenting to King Abdulaziz University Hospital with ischemic stroke (2017-2020), baseline NECT, and a visible defect on concurrent CTP. Using CTP as the reference standard, we measured the attenuation of ischemic and healthy contralateral brain on NECT to produce attenuation ratios (ischemic/normal) for penumbra and core. We used area under the receiver operating characteristic curve to estimate the optimal computed tomography (CT) attenuation ratio for penumbra. Per patient, we qualitatively assessed 8 regions within the affected cerebral hemisphere: on NECT as normal, hypoattenuating (with/out swelling), or isolated swelling and on CTP as normal, penumbra, or core. We sought associations between isolated swelling and penumbra, and between hypoattenuation and core., Results: We include 142 patients (86 male), mean age 61±14 years. Median 261 minutes (interquartile range, 173-382) to NECT. We measured 206 ischemic lesions (124 penumbra, 82 core). Optimal CT attenuation ratio for identifying penumbra was >0.87, with 86% sensitivity 91% specificity (area under the receiver operating characteristic curve, 0.95 [95% CI, 0.92-0.98]; P <0.0001). We qualitatively assessed 976 cerebral regions (72 isolated swelling, 254 hypoattenuation). On NECT, isolated swelling usually corresponded to CTP penumbra (70/72, 97%), whereas visible NECT hypoattenuation was found with core (141/254, 56%) and penumbra (109/254, 43%). CTP core lesions were rarely normal on NECT (13/155, 8%)., Conclusions: After ischemic stroke, brain tissue viability can be assessed using NECT. Isolated swelling is highly specific to penumbra. Visible hypoattenuation does not always represent core, nearly half of such lesions were penumbral on concurrent CTP and can be differentiated by measuring lesion attenuation.

Clinical outcomes of patients with acute ischemic stroke depend in part on the extent of their collateral circulation. A good collateral circulation has also been associated with greater benefit of intravenous thrombolysis and endovascular treatment. Treatment decisions for these reperfusion therapies are increasingly guided by a combination of clinical and imaging parameters, particularly in later time windows. Computed tomography and magnetic resonance imaging enable a rapid assessment of both the collateral extent and cerebral perfusion. Yet, the role of the collateral circulation in clinical decision-making is currently limited and may be underappreciated due to the use of rather coarse and rater-dependent grading methods. In this review, we discuss determinants of the collateral circulation in patients with acute ischemic stroke, report on commonly used and emerging neuroimaging techniques for assessing the collateral circulation, and discuss the therapeutic and prognostic implications of the collateral circulation in relation to reperfusion therapies for acute ischemic stroke.

Background: Tissue hypoxia plays a critical role in the events leading to cell death in ischemic stroke. Despite promising results in preclinical and small clinical pilot studies, inhaled oxygen supplementation has not translated to improved outcomes in large clinical trials. Moreover, clinical observations suggest that indiscriminate oxygen supplementation can adversely affect outcome, highlighting the need to develop novel approaches to selectively deliver oxygen to affected regions. This study tested the hypothesis that intravenous delivery of a novel oxygen carrier (Omniox-Ischemic Stroke [OMX-IS]), which selectively releases oxygen into severely ischemic tissue, could delay infarct progression in an established canine thromboembolic large vessel occlusion stroke model that replicates key dynamics of human infarct evolution., Methods: After endovascular placement of an autologous clot into the middle cerebral artery, animals received OMX-IS treatment or placebo 45 to 60 minutes after stroke onset. Perfusion-weighted magnetic resonance imaging was performed to define infarct progression dynamics to stratify animals into fast versus slow stroke evolvers. Serial diffusion-weighted magnetic resonance imaging was performed for up to 5 hours to quantify infarct evolution. Histology was performed postmortem to confirm final infarct size., Results: In fast evolvers, OMX-IS therapy substantially slowed infarct progression (by ≈1 hour, P <0.0001) and reduced the final normalized infarct volume as compared to controls (0.99 versus 0.88, control versus OMX-IS drug, P <0.0001). Among slow evolvers, OMX-IS treatment delayed infarct progression by approximately 45 minutes; however, this did not reach statistical significance ( P =0.09). The final normalized infarct volume also did not show a significant difference (0.93 versus 0.95, OMX-IS drug versus control, P =0.34). Postmortem histologically determined infarct volumes showed excellent concordance with the magnetic resonance imaging defined ischemic lesion volume (bias: 1.33% [95% CI, -15% to 18%)., Conclusions: Intravenous delivery of a novel oxygen carrier is a promising approach to delay infarct progression after ischemic stroke, especially in treating patients with large vessel occlusion stroke who cannot undergo definitive reperfusion therapy within a timely fashion.

Background and Purpose: Extracellular vesicles (EVs) are promising biomarkers for cerebral ischemic diseases, but not systematically tested in patients with transient ischemic attacks (TIAs). We aimed at (1) investigating the profile of EV-surface antigens in patients with symptoms suspicious for TIA; (2) developing and validating a predictive model for TIA diagnosis based on a specific EV-surface antigen profile., Methods: We analyzed 40 subjects with symptoms suspicious for TIA and 20 healthy controls from a training cohort. An independent cohort of 28 subjects served as external validation. Patients were stratified according to likelihood of having a real ischemic event using the Precise Diagnostic Score, defined as: unlikely (score 0–1), possible-probable (score 2–3), or very likely (score 4–8). Serum vesicles were quantified by nanoparticle tracking analysis and EV-surface antigen profile characterized by multiplex flow cytometry., Results: EV concentration increased in patients with very likely or possible-probable TIA (P<0.05) compared with controls. Nanoparticle concentration was directly correlated with the Precise Diagnostic score (R=0.712; P<0.001). After EV immuno-capturing, CD8, CD2, CD62P, melanoma-associated chondroitin sulfate proteoglycan, CD42a, CD44, CD326, CD142, CD31, and CD14 were identified as discriminants between groups. Receiver operating characteristic curve analysis confirmed a reliable diagnostic performance for each of these markers taken individually and for a compound marker derived from their linear combinations (area under the curve, 0.851). Finally, a random forest model combining the expression levels of selected markers achieved an accuracy of 96% and 78.9% for discriminating patients with a very likely TIA, in the training and external validation cohort, respectively., Conclusions: The EV-surface antigen profile appears to be different in patients with transient symptoms adjudicated to be very likely caused by brain ischemia compared with patients whose symptoms were less likely to due to brain ischemia. We propose an algorithm based on an EV-surface-antigen specific signature that might aid in the recognition of TIA.

Background and Purpose: Mechanical properties of thromboemboli play an important role in the efficacy of endovascular thrombectomy (EVT) for acute ischemic stroke. However, very limited data on mechanical properties of human stroke thrombi are available. We aimed to mechanically characterize thrombi retrieved with EVT, and to assess the relationship between thrombus composition and thrombus stiffness., Methods: Forty-one thrombi from 19 patients with acute stroke who underwent EVT between July and October 2019 were mechanically analyzed, directly after EVT. We performed unconfined compression experiments and determined tangent modulus at 75% strain (Et75) as a measure for thrombus stiffness. Thrombi were histologically analyzed for fibrin/platelets, erythrocytes, leukocytes, and platelets, and we assessed the relationship between histological components and Et75 with univariable and multivariable linear mixed regression., Results: Median Et75 was 560 (interquartile range, 393–1161) kPa. In the multivariable analysis, fibrin/platelets were associated with increased Et75 (aβ, 9 [95% CI, 5 to 13]) kPa, erythrocytes were associated with decreased Et75% (aβ, −9 [95% CI, −5 to −13]) kPa. We found no association between leukocytes and Et75. High platelet values were strongly associated with increased Et75 (aβ, 56 [95% CI, 38–73])., Conclusions: Fibrin/platelet content of thrombi retrieved with EVT for acute ischemic stroke is strongly associated with increased thrombus stiffness. For thrombi with high platelet values, there was a very strong relationship with thrombus stiffness. Our data provide a basis for future research on the development of next-generation EVT devices tailored to thrombus composition.

Background and Purpose: Brain atrophy can be regarded as an end-organ effect of cumulative cardiovascular risk factors. Accelerated brain atrophy is described following ischemic stroke, but it is not known whether atrophy rates vary over the poststroke period. Examining rates of brain atrophy allows the identification of potential therapeutic windows for interventions to prevent poststroke brain atrophy., Methods: We charted total and regional brain volume and cortical thickness trajectories, comparing atrophy rates over 2 time periods in the first year after ischemic stroke: within 3 months (early period) and between 3 and 12 months (later period). Patients with first-ever or recurrent ischemic stroke were recruited from 3 Melbourne hospitals at 1 of 2 poststroke time points: within 6 weeks (baseline) or 3 months. Whole-brain 3T magnetic resonance imaging was performed at 3 time points: baseline, 3 months, and 12 months. Eighty-six stroke participants completed testing at baseline; 125 at 3 months (76 baseline follow-up plus 49 delayed recruitment); and 113 participants at 12 months. Their data were compared with 40 healthy control participants with identical testing. We examined 5 brain measures: hippocampal volume, thalamic volume, total brain and hemispheric brain volume, and cortical thickness. We tested whether brain atrophy rates differed between time points and groups. A linear mixed-effect model was used to compare brain structural changes, including age, sex, years of education, a composite cerebrovascular risk factor score, and total intracranial volume as covariates., Results: Atrophy rates were greater in stroke than control participants. Ipsilesional hemispheric, hippocampal, and thalamic atrophy rates were 2 to 4 times greater in the early versus later period., Conclusions: Regional atrophy rates vary over the first year after stroke. Rapid brain volume loss in the first 3 months after stroke may represent a potential window for intervention. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02205424.

Background and Purpose: Ischemic infarction of the corpus callosum is rare and infarction isolated to the corpus callosum alone rarer still, accounting for much <1% of ischemic stroke in most stroke registries. About half of callosal infarctions affect the splenium., Methods: During a 2-week period, at the height of the coronavirus disease 2019 (COVID-19) pandemic in New York City, 4 patients at Montefiore Medical Center in the Bronx were found to have ischemic lesions of the splenium of the corpus callosum, 2 with infarction isolated to the corpus callosum., Results: All patients tested positive for COVID-19 and 3 had prolonged periods of intubation. All had cardiovascular risk factors. Clinically, all presented with encephalopathy and had evidence of coagulopathy and raised inflammatory markers., Conclusions: Infarction of the splenium of the corpus callosum is exceedingly rare and a cluster of such cases suggests COVID-19 as an inciting agent, with the mechanisms to be elucidated.

Background and Purpose: In ischemic stroke, inflammatory status may condition the development of collateral circulation. Here we assessed the relationship between systemic inflammatory biomarkers and collateral status in large vessel occlusion before mechanical thrombectomy., Methods: HIBISCUS-STROKE is a cohort study including acute ischemic stroke patients with large vessel occlusion treated with mechanical thrombectomy following admission magnetic resonance imaging. MMP-9 (matrix metalloproteinase-9) and MCP-1 (monocyte chemoattractant protein-1) were measured on blood sampling collected at admission. Collateral status was assessed on pretreatment Digital subtraction angiography and categorized into poor (Higashida score, 0-2) and good (Higashida score, 3-4). A multiple logistic regression model was performed to detect independent markers of good collateral status., Results: One hundred and twenty-two patients were included, of them 71 patients (58.2%) had a good collateral status. In univariate analysis, low MMP-9 levels ( P =0.01), high MCP-1 levels ( P <0.01), a low National Institute of Health Stroke Score ( P =0.046), a high diastolic blood pressure ( P =0.049), the absence of tandem occlusion ( P =0.046), a high Alberta Stroke Program Early CT Score ( P <0.01) and a low volume on the diffusion-weighted imaging ( P <0.01) were associated with good collateral status. Following multivariate analysis, low MMP-9 levels ( P =0.02) and high MCP-1 levels ( P <0.01) remained associated with good collateral status., Conclusions: Low MMP-9 and high MCP-1 levels were associated with good pretreatment collateral status in patients with acute ischemic stroke with large vessel occlusion. These results might suggest a relationship between collateral status and inflammation.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O 2 ) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O 2 ) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.

Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1 -deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T 2 weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T 2 weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading ( P <0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1 -deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.

Background and Purpose- Cerebral small vessel disease (SVD) is associated with increased stroke risk and poor stroke outcomes. We aimed to evaluate whether chronic SVD burden is associated with poor recruitment of collaterals in large-vessel occlusive stroke. Methods- Consecutive patients with middle cerebral artery or internal carotid artery occlusion presenting within 6 hours after stroke symptom onset who underwent thrombectomy from 2012 to 2017 were included. The prespecified primary outcome was poor collateral flow, which was assessed on baseline computed tomographic angiography (poor, ≤50% filling; good, >50% filling). Markers of chronic SVD on brain magnetic resonance imaging were rated for the extent of white matter hyperintensities, enlarged perivascular spaces, chronic lacunar infarctions and cerebral microbleeds using the Standards for Reporting Vascular Changes on Neuroimaging criteria. Severity of SVD was quantified by adding the presence of each SVD feature, with a total possible score of 0 to 4; each SVD type was also evaluated separately. Multivariable logistic regression analyses were performed to evaluate the relationships between SVD and poor collaterals, with adjustment for potential confounders. Results- Of the 100 eligible patients, the mean age was 65±16 years, median National Institutes of Health Stroke Scale score was 15, and 68% had any SVD. Poor collaterals were observed in 46%, and those with SVD were more likely to have poor collaterals than patients without SVD (aOR, 1.9 [95% CI, 1.1-3.2]). Of the SVD types, poor collaterals were significantly associated with white matter hyperintensities (aOR, 2.9 per Fazekas increment [95% CI, 1.6-5.3]) but not with enlarged perivascular spaces (adjusted odds ratio [aOR], 1.3 [95% CI, 0.4-4.0]), lacunae (aOR, 2.1 [95% CI, 0.6-7.1]), or cerebral microbleeds (aOR, 2.1 [95% CI, 0.6-7.8]). Having a greater number of different SVD markers was associated with a higher odds of poor collaterals (crude trend P <0.001; adjusted P =0.056). There was a dose-dependent relationship between white matter hyperintensity burden and poor collaterals: adjusted odds of poor collaterals were 1.5, 3.0, and 9.7 across Fazekas scores of 1 to 3 ( P trend=0.015). No patient with an SVD score of 4 had good collaterals. Conclusions- Chronic cerebral SVD is associated with poor recruitment of collaterals in large vessel occlusive stroke. A prospective study to elucidate the potential mechanism of how SVD may impair the recruitment of collaterals is ongoing.

Background and Purpose- Microglia/macrophages (Mi/MΦ) can profoundly influence stroke outcomes by acquiring functionally dominant phenotypes (proinflammatory or anti-inflammatory; deleterious or salutary). Identification of the molecular mechanisms that dictate the functional status of Mi/MΦ after brain ischemia/reperfusion may reveal novel therapeutic targets for stroke. We hypothesized that activation of TAK1 (transforming growth factor beta-activated kinase 1), a key MAP3K upstream of multiple inflammation-regulating pathways, drives Mi/MΦ toward a proinflammatory phenotype and potentiates ischemia/reperfusion brain injury. Methods- Young adult mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by reperfusion. TAK1 was targeted by tamoxifen-induced Mi/MΦ-specific knockout or administration of a selective inhibitor 5Z-7-Oxozeaenol after MCAO. Neurobehavioral deficits and long-term gray matter and white matter injury were assessed up to 35 days after MCAO. Mi/MΦ functional status and brain inflammatory profiles were assessed 3 days after MCAO by RNA-seq, flow cytometry, and immunohistochemistry. Results- TAK1 Mi/MΦ-specific knockout markedly ameliorated neurological deficits in the rotarod and cylinder tests for at least 35 days after MCAO. Mechanistically, RNA-seq of purified brain Mi/MΦ demonstrated that proinflammatory genes and their predicted biological functions were downregulated or inhibited in microglia and macrophages from TAK1 Mi/MΦ-specific knockout mice versus WT mice 3 days after MCAO. Consistent with the anti-inflammatory phenotype of Mi/MΦ-specific knockout, oxozeaenol treatment mitigated neuroinflammation 3 days after MCAO, manifested by less Iba1 + /CD16 + proinflammatory Mi/MΦ and suppressed brain invasion of various peripheral immune cells. Oxozeaenol treatment beginning 2 hours after MCAO improved long-term sensorimotor and cognitive functions in the foot fault, rotarod, and water maze tests. Furthermore, Oxozeaenol promoted both gray matter and white matter integrity 35 days after MCAO. Conclusions- TAK1 promotes ischemia/reperfusion-induced inflammation, brain injury, and maladaptive behavior by enhancing proinflammatory and deleterious Mi/MΦ responses. Therefore, TAK1 inhibition is a promising therapy to improve long-term stroke outcomes.

Background and Purpose- Phagocytic cells, such as microglia and blood-derived macrophages, are a key biological modality responsible for phagocytosis-mediated clearance of damaged, dead, or displaced cells that are compromised during senescence or pathological processes, including after stroke. This process of clearance is essential to eliminate the source of inflammation and to allow for optimal brain repair and functional recovery. Transcription factor, RXR (retinoic-X-receptor) is strongly implicated in phagocytic functions regulation, and as such could represent a novel target for brain recovery after stroke. Methods- Primary cultured microglia and bone marrow macrophages were used for phagocytic study. Mice with deleted RXR-α in myeloid phagocytes (Mac-RXR-α -/- ) were subjected to transient middle cerebral artery occlusion to mimic ischemic stroke and then treated with RXR agonist bexarotene. RNA-sequencing and long-term recovery were evaluated. Results- Using cultured microglia, we demonstrated that the RXR-α promotes the phagocytic functions of microglia toward apoptotic neurons. Using mice with deleted RXR-α in myeloid phagocytes (Mac-RXR-α -/- ), we have shown that despite behaving similarly to the control at early time points (up to 3 days, damage established histologically and behaviorally), these Mac-RXR-α -/- mice demonstrated worsened late functional recovery and developed brain atrophy that was larger in size than that seen in control mice. The RXR-α deficiency was associated with reduced expression of genes known to be under control of the prominent transcriptional RXR partner, PPAR (peroxisome proliferator-activated receptor)-γ, as well as genes encoding for scavenger receptors and genes that signify microglia/macrophages polarization to a reparative phenotype. Finally, we demonstrated that the RXR agonist, bexarotene, administered as late as 1 day after middle cerebral artery occlusion, improved neurological recovery, and reduced the atrophy volume as assessed 28 days after stroke. Bexarotene did not improve outcome in Mac-RXR-α -/- mice. Conclusions- Altogether, these data suggest that phagocytic cells control poststroke recovery and that RXR in these cells represents an attractive target with exceptionally long therapeutic window.

Background and Purpose- Blood-brain barrier (BBB) disruption is a critical pathological feature after stroke. MicroRNA-126 (miR-126) maintains BBB integrity by regulating endothelial cell function during development. However, the role of miR-126-3p and -5p in BBB integrity after stroke is unclear. Here, we investigated whether miR-126-3p and -5p overexpression regulates BBB integrity after cerebral ischemia. Methods- A lentivirus carrying genes encoding miR-126-3p or -5p was stereotactically injected into adult male Institute of Cancer Research mouse brains (n=36). Permanent middle cerebral artery occlusion was performed 2 weeks after virus injection. Brain infarct volume, edema volume, and modified neurological severity score were assessed at 1 and 3 days after ischemia. Immunostaining of ZO-1 (zonula occludens-1) and occludin was used to evaluate BBB integrity. IL-1β (interleukin-1β), TNF-α (tumor necrosis factor-α), VCAM-1 (vascular cell adhesion molecule-1), and E-selectin expression levels were determined by real-time polymerase chain reaction and Western blot analysis. Results- The expression of miR-126-3p and -5p decreased at 1 and 3 days after ischemia ( P <0.05). Injection of lentiviral miR-126-3p or -5p reduced brain infarct volume and edema volume ( P <0.05) and attenuated the decrease in ZO-1/occludin protein levels and IgG leakage at 3 days after stroke ( P <0.05). Injection of lentiviral miR-126-5p improved behavioral outcomes at 3 days after stroke ( P <0.05). miR-126-3p and -5p overexpression downregulated the expression of proinflammatory cytokines IL-1β and TNF-α and adhesion molecules VCAM-1 and E-selectin, as well as decreased MPO + (myeloperoxidase positive) cell numbers at 3 days after ischemia ( P <0.05). Conclusions- miR-126-3p and -5p overexpression reduced the expression of proinflammatory cytokines and adhesion molecules, and attenuated BBB disruption after ischemic stroke, suggesting that miR-126-3p and -5p are new therapeutic targets in the acute stage of stroke.

Background and Purpose- This study aims to investigate the association between the characteristics of atherosclerotic plaques of middle cerebral artery and recurrent ischemic stroke using magnetic resonance vessel wall imaging. Methods- One hundred and five patients with ischemic stroke attributed to middle cerebral artery plaque underwent high-resolution black-blood magnetic resonance vessel wall imaging. They were divided into group 1, with the first episode of acute stroke (imaging within 4 weeks of stroke, n=44); group 2, with recurrent acute stroke (n=29); and group 3, with chronic stroke (imaging after 3 months of stroke, n=32). Plaque characteristics including plaque area, plaque burden, contrast-enhancement ratio, eccentricity, and degree of stenosis were measured and compared across 3 groups. Association between plaque characteristics and recurrent strokes was investigated by multivariate analysis. Results- Plaque burden was significantly greater in recurrent stroke group than the other 2 groups (median: group 2, 82.7%, versus group 1, 76.3%, and group 3, 73.4%; P =0.001). Patients with acute stroke had higher enhancement ratio than patients with chronic stroke (median: group 1, 1.59, and group 2, 1.90, versus group 3, 1.33; P =0.014). Comparing to first-onset acute stroke patients, recurrent stroke patients were older, more likely with female sex and hypertension, and had higher plaque burden. After adjustment of clinical factors, plaque burden was the only independent imaging feature associated with recurrent stroke (odds ratio, 2.26, per 10% increase [95% CI, 1.03-4.96]; P =0.042). Conclusions- Higher plaque burden of middle cerebral artery identified on magnetic resonance vessel wall imaging is independently associated with recurrent ischemic stroke.

Background and Purpose- Low left ventricular ejection fraction (LVEF) leads to worse outcomes after stroke. We hypothesized that the arterial input function (AIF) variability on perfusion computed tomography, especially the time between scan onset and end of AIF (SO-EndAIF), would reflect reduction of cardiac output. Methods- Retrospective analysis of consecutive stroke patients, who underwent computed tomography between January 2013 and September 2018, was performed in 2 parts. (1) To determine the correlation between SO-EndAIF and LVEF, all patients with a transthoracic echocardiogram performed ±6 months from the time of stroke were included. LVEF was dichotomized as either normal (≥50%) or decreased (<50%). (2) AIF was compared with hypoperfusion volume, defined as delay time >3 seconds and with clinical outcome measured using 3-month modified Rankin Scale. Results- A total of 732 ischemic stroke patients underwent computed tomography, 231 with transthoracic echocardiogram were included in part (1), 393 with outcome data were included in part (2). In part (1), 193/231 (83.5%) had normal LVEF (median 61%) and 38/231 (16.5%) decreased LVEF (median 39%). The low-LVEF group had significantly prolonged SO-EndAIF compared with normal-LVEF group (mean of 39.7 versus 26 second; P <0.001), and larger hypoperfusion lesions (94.9 versus 37.6 mL; P <0.001). SO-EndAIF time was strongly associated with EF, with an area under the curve of 0.86. Twenty nine seconds was the best threshold to distinguish between normal and impaired EF (area under the curve, 0.77). In part (2), the SO-EndAIF ≥29 second group had larger hypoperfusion volumes (21.8 versus 89.7 mL; P <0.001) and infarct core (12.2 versus 2.3 mL; P <0.0001) and patients with SO-EndAIF ≥29 seconds had fewer excellent or good clinical outcomes (modified Rankin Scale score 0-1; 40% versus 22%; OR, 2.79; P <0.001, modified Rankin Scale score 0-2; 65% versus 35%; OR, 1.41; P =0.033). Conclusions- AIF width correlates with ejection fraction in acute ischemic stroke. A 29-second threshold from scan onset to end of AIF accurately predicts reduced LVEF and identifies patients more likely to have worse outcomes after stroke.

Background and Purpose- To test the hypothesis that covert brain infarcts (CBIs) are more likely to be located in noneloquent brain areas compared with clinical strokes and that CBI etiological subtypes carry a differential risk of vascular events compared with people without CBI. Methods- We used brain magnetic resonance imaging from 1290 stroke-free participants in the NOMAS (Northern Manhattan Study) to evaluate for CBI. We classified CBI as cardioembolic (ie, known atrial fibrillation), large artery atherosclerosis (extracranial and intracranial), penetrating artery disease, and cryptogenic (no apparent cause). CBI localized in the nonmotor areas of the right hemisphere were considered noneloquent. We then evaluated risk of events by CBI subtype with adjusted Cox proportional models. Results- At the time of magnetic resonance imaging, 236 participants (18%) had CBI (144 [61%] distal cryptogenic, 29 [12%] distal cardioembolic, 26 [11%] large artery atherosclerosis, and 37 [16%] penetrating artery disease). Smaller (per mm, odds ratio, 0.8 [0.8-0.9]) and nonbrain stem infarcts (odds ratio, 0.2 [0.1-0.6]) were more likely to be covert. During the follow-up period (10.4±3.1 years), 398 (31%) died (162 [13%] of vascular death) and 117 (9%) had a stroke (99 [85%]) were ischemic. Risks of events varied by CBI subtype, with the highest risk of stroke (hazard ratio, 2.2 [1.3-3.7]) and vascular death (hazard ratio, 2.24 [1.29-3.88]) noted in participants with intracranial large artery atherosclerosis-related CBI. Conclusions- CBI can be classified into subtypes that have differential outcomes. Certain CBI subtypes such as those related to intracranial large artery atherosclerosis have a high risk of adverse vascular outcomes and could warrant consideration of treatment trials.

Background and Purpose- Hypertension is the most frequent comorbidity in stroke.The purpose of this study was to evaluate whether hypertension alters the response to treatment with adipose tissue-derived mesenchymal stem cells (ADMSCs) after an ischemic stroke in rats. Methods- Ischemic stroke was induced in male normotensive or hypertensive rats. Either vehicle or 1×10 6 ADMSC was intravenously administered at 48 hours poststroke. Functional outcome, lesion size and volume, and markers of brain repair (GFAP [glial fibrillary acidic protein], doublecortin, CD-31, α-smooth muscle actin) were evaluated. Results- Hypertensive rats had larger lesions, higher apparent diffusion coefficients (ADC) and worse functional outcomes than normotensive rats. Hypertension increased GFAP and vascular markers (CD-31 and α-smooth muscle actin). The hypertensive rats treated with ADMSC did not show any significant improvement in functional recovery, lesion size, ADC values, or histological markers compared with those which received the vehicle. Conclusions- ADMSC did not reverse the hypertension-induced increase in lesion severity or functional impairment. Gliosis, neurogenesis, or vascular markers were not affected by ADMSC in hypertensive rats. Hypertension has a negative impact on the therapeutic effect of ADMSC after an ischemic stroke.

Background and Purpose- Poor collateral flow is associated with poor clinical outcome in acute ischemic stroke and may indicate futile recanalization after successful thrombectomy. Pronounced early formation of cerebral ischemic edema may be the link between poor collateral status and declined functional outcome, but this relationship has not been investigated yet. We hypothesized that collateral status is associated with early lesion water uptake as quantitative marker for edema progression. Methods- One hundred seventy-six patients with middle cerebral artery stroke who underwent mechanical thrombectomy were analyzed. Status of cerebral collateral circulation (collaterals status [CS]) was derived using an established 5-point scoring system in admission computed tomography angiography, and good collaterals were defined as CS 3 to 4. Ischemic brain edema dynamics were quantified using early edema progression rate (EPR). EPR was derived from quantitative lesion water uptake in admission computed tomography divided by time from symptom onset to imaging. Good clinical outcome was defined as modified Rankin Scale score 0 to 2 after 90 days. Results- The median EPR was 1.4% per hour (interquartile range, 0.5-3.5%) in patients with good collaterals, which was lower than the median EPR in patients with poor collaterals of 5.8% per hour (interquartile range, 2.1-5.9%; P <0.0001). In multivariable regression analysis, lower CS was significantly and independently associated with higher EPR (1.6% EPR per 1-point CS; P =0.002). A higher EPR was associated with reduced likelihood of good clinical outcome: odds ratio 0.87; (95% CI, 0.76-0.99; P =0.03). Conclusions- Patients with poor CS had significantly higher EPR, which was associated with worse clinical outcome. These patients might benefit from adjuvant antiedematous treatment.

Background and Purpose- Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca 2+ entry, which is triggered by the depletion of intracellular calcium stores, contributes to ischemia-induced calcium influx in neurons, but the responsible Ca 2+ channel is not known. Methods- Here, we have generated mice lacking the calcium channel subunit Orai2 and analyzed them in experimental stroke. Results- Orai2-deficient mice were protected from ischemic neuronal death both during acute ischemia under vessel occlusion and during ischemia/reperfusion upon successful recanalization. Calcium signals induced by calcium store depletion or oxygen/glucose deprivation were significantly diminished in Orai2-deficient neurons demonstrating that Orai2 is a central mediator of neuronal capacitative Ca 2+ entry and is involved in calcium overload during ischemia. Conclusions- Our experimental data identify Orai2 as an attractive target for pharmaceutical intervention in acute stroke.

Background and Purpose- Standard analytic approaches (eg, logistic regression) fail to adequately control for time-dependent confounding and, therefore, may yield biased estimates of the total effect of the exposure on the outcome. In the present study, we estimate the effect of body mass index, intentional physical activity, HDL (high-density lipoprotein) cholesterol, LDL (low-density lipoprotein) cholesterol, hypertension, and cigarette smoking on the 11-year risk of ischemic stroke by sex using the parametric g-formula to control time-dependent confounders. Methods- Using data from the MESA (Multi-Ethnic Study of Atherosclerosis), we followed 6809 men and women aged 45 to 84 years. We estimated the risk of stroke under 6 hypothetical interventions: maintaining body mass index <25 kg/m 2 , maintaining normotension (systolic blood pressure <140 and diastolic <90 mm Hg), quitting smoking, maintaining HDL >1.55 mmol/L, maintaining LDL <3.11 mmol/L, and exercising at least 210 minutes per week. The effects of joint hypothetical interventions were also simulated. Results- In men, the 11-year risk of ischemic stroke would be reduced by 85% (95% CI, 66-96) for all 6 hypothetical interventions. In women, this same effect was estimated as 55% (95% CI, 6-82). Conclusions- The hypothetical interventions explored in our study resulted in risk reduction in both men and women.

Background and Purpose- Brain electrical stimulation, widely studied to facilitate recovery from stroke, has also been reported to confer direct neuroprotection in preclinical models of acute cerebral ischemia. Systematic review of controlled preclinical acute cerebral ischemia studies would aid in planning for initial human clinical trials. Methods- A systematic Medline search identified controlled, preclinical studies of central nervous system electrical stimulation in acute cerebral ischemia. Studies were categorized among 6 stimulation strategies. Three strategies applied different stimulation types to tissues within the ischemic zone (cathodal hemispheric stimulation [CHS], anodal hemispheric stimulation, and pulsed hemispheric stimulation), and 3 strategies applied deep brain stimulation to different neuronal targets remote from the ischemic zone (fastigial nucleus stimulation, subthalamic vasodilator area stimulation, and dorsal periaqueductal gray stimulation). Random-effects meta-analysis assessed electrical stimulation modification of final infarct volume. Study-level risk of bias and intervention-level readiness-for-translation were assessed using formal rating scales. Results- Systematic search identified 28 experiments in 21 studies, including a total of 350 animals, of electrical stimulation in preclinical acute cerebral ischemia. Overall, in animals undergoing electrical stimulation, final infarct volumes were reduced by 37% (95% CI, 34%-40%; P <0.001), compared with control. There was evidence of heterogeneity of efficacy among stimulation strategies ( I 2 =93.1%, P heterogeneity <0.001). Among the within-ischemic zone stimulation strategies, only CHS significantly reduced the infarct volume (27 %; 95% CI, 22%-33%; P <0.001); among the remote-from ischemic zone approaches, all (fastigial nucleus stimulation, subthalamic vasodilator area stimulation, and dorsal periaqueductal gray stimulation) reduced infarct volumes by approximately half. On formal rating scales, CHS studies had the lowest risk of bias, and CHS had the highest overall quality of intervention-level evidence supporting readiness to proceed to clinical testing. Conclusions- Electrical stimulation reduces final infarct volume across preclinical studies. CHS shows the most robust evidence and is potentially appropriate for progression to early-stage human clinical trial testing as a promising neuroprotective intervention.

Background and Purpose- Early arterial recanalization in acute ischemic stroke is strongly associated with better outcomes. However, early worsening of arterial patency was seldom studied. We investigated potential predictors and long-term prognosis of worsening of arterial patency at 24 hours after stroke onset. Methods- Patients from the Acute Stroke Registry and Analysis of Lausanne registry including admission and 24-hour vascular imaging (computed tomography or magnetic resonance angiography) were included. Worsening of arterial patency was defined as a new occlusion and significant stenosis in any extracranial or intracranial artery, comparing 24 hours with admission imaging. Variables associated with worsening of arterial patency were assessed by stepwise multiple logistic regression. The impact of arterial worsening on 3-month outcome was investigated with an adjusted modified Rankin Scale shift analysis. Results- Among 2152 included patients, 1387 (64.5%) received intravenous thrombolysis and endovascular treatment, and 65 (3.0%) experienced 24-hour worsening of arterial patency. In multivariable analysis, history of hypertension seemed protective (adjusted odds ratio [aOR], 0.45; 95% CI, 0.27-0.75) while higher admission National Institutes of Health Stroke Scale (aOR, 1.06; 95% CI, 1.02-1.10), intracranial (aOR, 4.78; 95% CI, 2.03-11.25) and extracranial stenosis (aOR, 3.67; 95% CI, 1.95-6.93), and good collaterals (aOR, 3.71; 95% CI, 1.54-8.95) were independent predictors of worsening of arterial patency. Its occurrence was associated with a major unfavorable shift in the distribution of the modified Rankin Scale at 3 months (aOR, 5.97; 95% CI, 3.64-9.79). Conclusions- Stroke severity and admission vascular imaging findings may help to identify patients at a higher risk of developing worsening of arterial patency at 24 hours. The impact of worsening of arterial patency on long-term outcome warrants better methods to detect and prevent this early complication.

Background and Purpose- Sex differences in the incidence and outcome of stroke have been well documented. The severity of stroke in women is, in general, significantly lower than that in men, which is mediated, at least in part, by the protective effects of β-estradiol. However, the detailed mechanisms underlying the neuroprotection by β-estradiol are still elusive. Recent studies have demonstrated that activation of ASIC1a (acid-sensing ion channel 1a) by tissue acidosis, a common feature of brain ischemia, plays an important role in ischemic brain injury. In the present study, we assessed the effects of β-estradiol on acidosis-mediated and ischemic neuronal injury both in vitro and in vivo and explored the involvement of ASIC1a and underlying mechanism. Methods- Cultured neurons and NS20Y cells were subjected to acidosis-mediated injury in vitro. Cell viability and cytotoxicity were measured by methylthiazolyldiphenyl-tetrazolium bromide and lactate dehydrogenase assays, respectively. Transient (60 minutes) focal ischemia in mice was induced by suture occlusion of the middle cerebral artery in vivo. ASIC currents were recorded using whole-cell patch-clamp technique while intracellular Ca 2+ concentration was measured with fluorescence imaging using Fura-2. ASIC1a expression was detected by Western blotting and quantitative real-time polymerase chain reaction. Results- Treatment of neuronal cells with β-estradiol decreased acidosis-induced cytotoxicity. ASIC currents and acid-induced elevation of intracellular Ca 2+ were all attenuated by β-estradiol treatment. In addition, we showed that β-estradiol treatment reduced ASIC1a protein expression, which was mediated by increased protein degradation, and that estrogen receptor α was involved. Finally, we showed that the level of ASIC1a protein expression in brain tissues and the degree of neuroprotection by ASIC1a blockade were lower in female mice, which could be attenuated by ovariectomy. Conclusions- β-estradiol can protect neurons against acidosis-mediated neurotoxicity and ischemic brain injury by suppressing ASIC1a protein expression and channel function. Visual Overview- An online visual overview is available for this article.

Background and Purpose- In patients with symptomatic intracranial atherosclerotic stenosis, identifying the underlying stroke mechanisms may inform secondary prevention. We aimed to propose reproducible classification criteria for stroke mechanisms based on routine neuroimaging in symptomatic intracranial atherosclerotic stenosis and explore their clinical implications. Methods- We recruited patients with acute ischemic stroke attributed to 50% to 99% intracranial atherosclerotic stenosis in anterior circulation from 2 centers. Two investigators independently classified probable stroke mechanisms as parent artery atherosclerosis occluding penetrating artery, artery-to-artery embolism, hypoperfusion, and mixed mechanisms, with prespecified criteria based on infarct topography and magnetic resonance/computed tomography angiography. These stroke mechanisms were correlated with features of the patients at baseline and recurrent ischemic stroke in the same territory or relevant transient ischemic attack within 1 year. Results- Among 153 patients recruited, the most common stroke mechanisms were isolated hypoperfusion (35.3%) and mixed mechanism of artery-to-artery embolism and hypoperfusion (37.3%) that was associated with higher incidence of dyslipidemia ( P =0.045) and hypertension ( P =0.033) than patients with other stroke mechanisms. The proposed criteria showed substantial to excellent intrarater and interrater reproducibilities (κ, 0.791-0.908). Overall, 31 patients received interventional treatment of the diseased intracranial artery; 122 received medical treatment, among whom a mixed mechanism of artery-to-artery embolism and hypoperfusion at baseline was associated with higher risk of ischemic stroke in the same territory within 1 year (24.4% versus 7.8%; hazard ratio, 3.40; 95% CI, 1.25-9.20; log-rank P =0.010) than other mechanisms combined. Conclusions- Artery-to-artery embolism and hypoperfusion commonly coexist in ischemic stroke attributed to intracranial atherosclerotic stenosis, which may be associated with higher risk of stroke relapse.

Background and Purpose- After ischemic injury, microglia and infiltrated macrophages may acquire different polarization phenotypes promoting inflammation and injury (M1) or repair and protection (M2). There is evidence that immunomodulation, via type 2 helper T-cells (Th2) cytokines, exerts neuroprotection after ischemia. We investigated the consequences of simultaneous genetic deletion of Th2 cytokines (IL [interleukin]-4, IL-5, IL-9, IL-13) on the histopathologic outcome, microglia and infiltrated macrophages markers, and ischemic microenvironment at different time points after ischemic injury in mice subjected to permanent occlusion of the middle cerebral artery. Methods- Wild-type and Th2 cytokine-deficient mice (4KO) were subjected to permanent occlusion of the middle cerebral artery by electrocoagulation and followed up to 5 weeks after permanent occlusion of the middle cerebral artery. Neuropathologic outcome was assessed at 24 hours (n=6), 7 days (n=6), and 5 weeks (n=6-7) by examination of the ischemic lesion, neuronal count, microglia and infiltrated macrophages markers, brain atrophy, collagen deposition, and GFAP (glial fibrillary acidic protein) immunohistochemistry. Selected gene expression was investigated at 7 days (n=6). Results- 4KO mice showed no difference in lesion and neuronal count 7 days and up to 5 weeks after permanent occlusion of the middle cerebral artery compared with wild type. Ischemic 4KO mice had lower CD16/32 expression at 24 hours, lower CD11b and CD16/32 expression at 7 days than wild type. They had higher CD206 expression at 24 hours, higher CD206 and arginase1 at 7 days, and increased mRNA for CXCL9 (chemokine [C-X-C motif] ligand 9) compared with wild type. Additional histopathologic analysis, including brain atrophy, gliotic scar, and collagenous scar confirmed no difference between genotypes at 5 weeks. Conclusions- This study casts light on the proposed neuroprotective function of Th2 cytokines, showing that combined IL-4, IL-5, IL-9, IL-13 deletion does not affect the neuropathologic response to ischemic stroke in the subacute and chronic phases. Our findings indicate that Th2 cytokines are not an essential neuroimmunological cue able to drive the brain's ischemic outcome.

Background and Purpose- Sex differences in the management and outcomes of acute ischemic stroke with large vessel occlusion are unknown in the era of endovascular therapy (EVT). This study investigated these differences in the RESCUE (Recovery by Endovascular Salvage for Cerebral Ultra-Acute Embolism)-Japan Registry 2 patient database. Methods- RESCUE-Japan Registry 2 registered patients with large vessel occlusion who were admitted within 24 hours of onset were enrolled in this study. We estimated the likelihood to receive EVT according to sex. The primary outcome was good outcome defined as a modified Rankin Scale score of 0 to 2 at 90 days after onset. Secondary outcomes were mortality within 90 days, any or symptomatic intracranial hemorrhage within 72 hours, and recurrence of stroke or transient ischemic attack within 90 days. Results- Among 2399 patients, 1087 patients were female and 1312 were male; 47.9% of females and 57.7% of males received EVT (adjusted odds ratio, 0.71; 95% CI, 0.59-0.86). Good outcome was observed in 27.3% and 44.2% of the females and males, respectively ( P<0.0001). The adjusted odds ratio of a good outcome in females was 0.80 (95% CI, 0.65-0.99). Mortality was 12.3% and 9.9% in females and males, respectively ( P=0.06); the adjusted odds ratio was 0.78 (95% CI, 0.58-1.05). Conclusions- Females with acute ischemic stroke with large vessel occlusion showed poor functional outcome compared to males. Females were less likely to receive EVT; lower utilization of EVT accounted for a portion of the poor outcome.

Background and Purpose- Ischemic stroke is one of the leading causes of morbidity and mortality worldwide and a major cause of long-term disability. Recently, long noncoding RNAs have been revealed, which are tightly associated with several human diseases. However, the functions of long noncoding RNAs in ischemic stroke still remain largely unknown. In the current study, for the first time, we investigated the role of long noncoding RNA Nespas in ischemic stroke. Methods- We used in vivo models of middle cerebral artery occlusion and in vitro models of oxygen-glucose deprivation to illustrate the effect of long noncoding RNA Nespas on ischemic stroke. Results- We found expression of Nespas was significantly increased in ischemic cerebral tissues and oxygen-glucose deprivation-treated BV2 cells in a time-dependent manner. Silencing of Nespas aggravated middle cerebral artery occlusion operation-induced IR injury and cell death. In addition, proinflammatory cytokine production and NF-κB (nuclear factor-κB) signaling activation were inhibited by Nespas overexpression. TAK1 (transforming growth factor-β-activated kinase 1) was found to directly interact with Nespas, and TAK1 activation was significantly suppressed by Nespas. At last, we found Nespas-inhibited TRIM8 (tripartite motif 8)-induced K63-linked polyubiquitination of TAK1. Conclusions- We showed that Nespas played anti-inflammatory and antiapoptotic roles in cultured microglial cells after oxygen-glucose deprivation stimulation and in mice after ischemic stroke by inhibiting TRIM8-related K63-linked polyubiquitination of TAK1.

Background and Purpose- Ischemic stroke, a complex and heterogeneous disease, is the second leading cause of death worldwide. Genetic factors and epigenetic modification contribute to the pathogenesis of this disease. However, the effects of epigenetic factors on this disease have not been systematically investigated. Our study was designed to identify methylation alterations in large-artery atherosclerotic stroke. Methods- We conducted an epigenome-wide association analysis of large-artery atherosclerotic stroke using an Infinium HumanMethylation450 array (cases:controls=12:12), and the differentially methylated loci were validated in 2 cohorts (cases:controls, 110:122 and 191:191, respectively) using a Sequenom EpiTYPER assay. Results- In the screening stage, 1012 differentially methylated CpG sites annotated in 672 genes were found to be significantly associated with large-artery atherosclerotic stroke (mean methylation difference >5%, P<0.01). Disease, Gene Ontology, and pathway analysis highlighted the enrichment of these differentially methylated genes in cardiovascular, metabolic, neurological and immune-related functional gene clusters ( P<0.05). We identified a differentially methylated region in the promoter of a humanin gene ( MTRNR2L8, mean methylation difference=-13.01%, P=8.86×10 -14 ). We constructed a diagnostic prediction model that was based on the mean number of significantly changed CpG loci in MTRNR2L8 and showed high diagnostic specificity and sensitivity ( P<0.0001, area under the curve=0.774). Conclusions- Together, these findings demonstrate that DNA methylation plays an important role in large-artery atherosclerotic stroke and that methylation of MTRNR2L8 is a potential therapeutic target and diagnostic biomarker for stroke.

Background and Purpose- A major process contributing to cell death in the ischemic brain is inflammation. Inflammasomes are multimolecular protein complexes that drive inflammation through activation of proinflammatory cytokines, such as IL (interleukin)-1β. Preclinical evidence suggests that IL-1β contributes to a worsening of ischemic brain injury. Methods- Using a mouse middle cerebral artery thrombosis model, we examined the inflammatory response after stroke and the contribution of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome to ischemic injury. Results- There was a marked inflammatory response after stroke characterized by increased expression of proinflammatory cytokines and NLRP3 and by recruitment of leukocytes to the injured tissue. Targeting NLRP3 with the inhibitor MCC950, or using mice in which NLRP3 was knocked out, had no effect on the extent of injury caused by stroke. Conclusions- These data suggest that the NLRP3 pathway does not contribute to the inflammation exacerbating ischemic brain damage, contradicting several recent reports to the contrary.

Background and Purpose- Inhibition of brain NKCC1 (Na + -K + -Cl - cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Background and Purpose- Recent guidelines have suggested the potential benefit of intravenous thrombolysis in stroke patients with systemic malignancy who have a reasonable life expectancy of >6 months. However, it is difficult to determine which patients with cancer will have a life expectancy of >6 months. Therefore, we identified the factors associated with 6-month mortality in patients with acute ischemic stroke and systemic malignancy. Methods- Consecutive stroke patients with systemic malignancy were retrospectively analyzed. We classified the patients into 3 groups: the nonactive cancer, active nonmetastatic cancer, and metastatic cancer groups. We compared the baseline characteristics and 6-month survival rates. Results- Of the 468 ischemic stroke patients with systemic malignancy during an 8-year period, 223 patients had nonactive cancer, 105 patients had active nonmetastatic cancer, and 140 patients had metastasis. During the 6-month follow-up, 122 patients (26.1%) died (nonactive cancer group [7.2%, 16/223], active nonmetastatic cancer group [11.4%, 12/105], and metastatic cancer group [67.1%, 94/140]). Multivariate Cox regression analysis revealed that the presence of metastasis (hazard ratio, 4.527; 95% CI, 2.175-9.422) was independently associated with 6-month mortality. However, the active nonmetastatic cancer group exhibited similar 6-month mortality to the nonactive cancer group (hazard ratio, 0.711; 95% CI, 0.282-1.795). Gastric/esophageal cancer and pancreatic cancer were also independently associated with 6-month mortality (hazard ratio, 2.068 and 2.389, respectively). Conclusions- In stroke patients with active cancer, the presence of metastasis and the cancer type were crucial factors associated with 6-month mortality.

Background and Purpose- Acetylsalicylic acid and clopidogrel are the 2 main antithrombotic drugs for secondary prevention in patients with ischemic stroke (IS) without indication for anticoagulation. Because of their limited efficacy and potential side effects, novel antiplatelet agents are urgently needed. Cilostazol, a specific phosphodiesterase (PDE)-3 inhibitor, protected from IS in clinical studies comprising mainly Asian populations. Nevertheless, the detailed mechanistic role of PDE-3 inhibitors in IS pathophysiology is hardly understood. In this project, we analyzed the efficacy and pathophysiologic mechanisms of a novel and only recently described PDE-3 inhibitor (substance V) in a mouse model of focal cerebral ischemia. Methods- Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in 6- to 8-week-old male C57Bl/6 wild-type mice receiving substance V or vehicle 1 hour after ischemia induction. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, as well as the extent of local inflammatory response and cell death, was determined. Results- Inhibition of PDE-3 by pharmacological blockade with substance V significantly reduced infarct volumes and improved neurological outcome on day 1 and 7 after experimental cerebral ischemia. Reduced blood-brain barrier damage, attenuated brain tissue inflammation, and decreased local cell death could be identified as potential mechanisms. PDE-3 inhibitor treatment did neither increase the number of intracerebral hemorrhages nor affect platelet function. Conclusions- The novel PDE-3 inhibitor substance V protected mice from IS independent from platelet function. Pharmaceutical inactivation of PDE-3 might become a promising therapeutic approach to combat IS via inhibition of thromboinflammatory mechanisms and stabilization of the blood-brain barrier.

Background and Purpose- In the setting of acute ischemic stroke because of large-vessel occlusion (LVO) there is progressive loss of brain tissue which occurs in a time-dependent fashion previously quantified to be ≈1.9 million neurons per minute. However, this number represents an average and accumulating evidence suggests large individual variation. In this study, we aim to quantify the distribution and range in the rate of loss brain tissue across the entire spectrum of clinical phenotypes of anterior circulation LVO strokes encountered in clinical practice. Methods- Retrospective review of a prospectively acquired database of consecutive patients with anterior circulation stroke because of proximal LVO and appropriate ischemic core imaging was performed. Ischemic core volume was measured using automated software processing and time from last known well to imaging was recorded. Applying previously published methodology for brain loss quantification, we computed rate of brain tissue elements loss in proximal LVO stroke patients. Results- We studied 415 patients with internal carotid artery or middle cerebral artery (M1 segment) occlusion. Mean ischemic core volume was 50.4 mL and mean time to imaging from time from last known well (TLKW) was 8.7 hours, which is similar to previously published data, translates into a mean loss per minute of 2.03 million neurons, 14.8 billion synapses, and 12.8 km of myelinated fibers. However, the distribution of neuron loss was highly variable, ranging from <35 000 to >27 million cells per minute. Conclusions- Widely spread rates of infarct growth are observed in acute ischemic stroke because of proximal LVO with rate of neuron loss per minute ranging from <35 000 per minute in slow progressors to >27 million per minute in fast progressors, with a mean and median of 2 million and 0.9 million, respectively.

Background and Purpose- The volume of estimated ischemic core using computed tomography perfusion (CTP) imaging can identify ischemic stroke patients who are likely to benefit from reperfusion, particularly beyond standard time windows. We assessed the accuracy of pretreatment CTP estimated ischemic core in patients with successful endovascular reperfusion. Methods- Patients from the HERMES (Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials) and EXTEND-IA TNK (Tenecteplase Versus Alteplase Before Endovascular Therapy for Ischemic Stroke) databases who had pretreatment CTP, >50% angiographic reperfusion, and follow-up magnetic resonance imaging at 24 hours were included. Ischemic core volume on baseline CTP data was estimated using relative cerebral blood flow <30% (RAPID, iSchemaView). Follow-up diffusion magnetic resonance imaging was registered to CTP, and the diffusion lesion was outlined using a semiautomated algorithm. Volumetric and spatial agreement (using Dice similarity coefficient, average Hausdorff distance, and precision) was assessed, and expert visual assessment of quality was performed. Results- In 120 patients, median CTP estimated ischemic core volume was 7.8 mL (IQR, 1.8-19.9 mL), and median diffusion lesion volume at 24 hours was 30.8 mL (IQR, 14.9-67.6 mL). Median volumetric difference was 4.4 mL (IQR, 1.2-12.0 mL). Dice similarity coefficient was low (median, 0.24; IQR, 0.15-0.37). The median precision (positive predictive value) of 0.68 (IQR, 0.40-0.88) and average Hausdorff distance (median, 3.1; IQR, 1.8-5.7 mm) indicated reasonable spatial agreement for regions estimated as ischemic core at baseline. Overestimation of total ischemic core volume by CTP was uncommon. Expert visual review revealed overestimation predominantly in white matter regions. Conclusions- CTP estimated ischemic core volumes were substantially smaller than follow-up diffusion-weighted imaging lesions at 24 hours despite endovascular reperfusion within 2 hours of imaging. This may be partly because of infarct growth. Volumetric CTP core overestimation was uncommon and not related to imaging-to-reperfusion time. Core overestimation in white matter should be a focus of future efforts to improve CTP accuracy.

Background and Purpose- Type 2 diabetes mellitus (T2DM) is a major comorbidity that exacerbates ischemic brain injury and worsens functional outcome after stroke. T2DM is known to aggravate white matter (WM) impairment, but the underlying mechanism is not completely understood. This study was designed to test the hypothesis that T2DM impedes poststroke WM recovery by suppressing both oligodendrogenesis and beneficial microglia/macrophage responses. Methods- Permanent distal middle cerebral artery occlusion was performed in wild-type, homozygous diabetic db/db, and heterozygous db/+ mice. The adhesive removal, open field, and Morris water maze tests were used to assess neurobehavioral outcomes. Neuronal tissue loss, WM damage, oligodendrogenesis, and microglia/macrophage responses were evaluated up to 35 days after stroke. The functional integrity of WM was measured by electrophysiology. Primary microglia-oligodendrocyte cocultures were used for additional mechanistic studies. Results- T2DM exacerbated structural damage and impaired conduction of compound action potentials in WM 35 days after stroke. The deterioration in WM integrity correlated with poor sensorimotor performance. Furthermore, T2DM impaired the proliferation of oligodendrocyte precursor cells and the generation of new myelinating oligodendrocytes. T2DM also promoted a shift of microglia/macrophage phenotype toward the proinflammatory modality. Coculture studies confirmed that microglia/macrophage polarization toward the proinflammatory phenotype under high glucose conditions suppressed oligodendrocyte precursor cell differentiation. Conclusions- Deterioration of WM integrity and impairments in oligodendrogenesis after stroke are associated with poor long-term functional outcomes in experimental diabetes mellitus. High glucose concentrations may shift microglia/macrophage polarization toward a proinflammatory phenotype, significantly impairing oligodendrocyte precursor cell differentiation and WM repair.

Background and Purpose- We aimed to compare the ability of conventional Alberta Stroke Program Early CT Score (ASPECTS), automated ASPECTS, and ischemic core volume on computed tomographic perfusion to predict clinical outcome in ischemic stroke because of large vessel occlusion ≤18 hours after symptom onset. Methods- We selected patients with acute ischemic stroke from the CRISP study (Computed Tomographic Perfusion to Predict Response to Recanalization in Ischemic Stroke Project) with successful reperfusion (modified treatment in cerebral ischemia score 2b or 3). We used e-ASPECTS software to calculate automated ASPECTS and RAPID software to estimate ischemic core volumes. We studied associations between these imaging characteristics and good outcome (modified Rankin Scale score, 0-2) or poor outcome (modified Rankin Scale score, 4-6) in univariable and multivariable analysis, after adjustment for relevant clinical confounders. Results- We included 156 patients. Conventional and automated ASPECTS was not associated with good or poor outcome in univariable analysis ( P=nonsignificant for all). Automated ASPECTS was associated with good outcome in multivariable analysis ( P=0.02) but not with poor outcome. Ischemic core volume was associated with good ( P<0.01) and poor outcome ( P=0.04) in univariable and multivariable analysis ( P=0.03 and P=0.02, respectively). Computed tomographic perfusion predicted good outcome with an area under the curve of 0.62 (95% CI, 0.53-0.71) and optimal cutoff core volume of 15 mL. Conclusions- Ischemic core volume assessed on computed tomographic perfusion is a predictor of clinical outcome among patients in whom endovascular reperfusion is achieved ≤18 hours after symptom onset. In this population, conventional or automated ASPECTS did not predict outcome.

Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body β-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Background and Purpose: Detection and localization of the early phase of blood-brain barrier disruption (BBBD) in vivo during cerebral ischemia/reperfusion injury remain a major challenge but may be a relevant outcome parameter in stroke., Methods: We studied early BBBD in mice after transient middle cerebral artery occlusion by multimodal, high-field (9.4T) in vivo magnetic resonance imaging, including the contrast agent gadofluorineM as an albumin-binding tracer. GadofluorineM contrast-enhanced magnetic resonance imaging was performed to determine BBBD at 2, 6, and 24 hours after reperfusion. BBBD was confirmed and localized along the microvascular tree by using fluorescent gadofluorineM and immunofluorescence stainings (cluster of differentiation 31, ephrin type-B receptor 4, alpha smooth muscle actin, ionized calcium binding adaptor molecule 1)., Results: GadofluorineM contrast-enhanced magnetic resonance imaging revealed a multifocal spatial distribution of early BBBD and its close association with the microvasculature at a resolution of 40 μm. GadofluorineM leakage was closely associated with ephrin type-B receptor 4-positive but not alpha smooth muscle actin-positive vessels. The multifocal pattern of early BBBD (already at 2 hours after reperfusion) thus occurred in the distal capillary and venular microvascular bed. These multifocal zones showed distinct imaging signs indicative of early vasogenic edema. The total volume of multifocal early BBBD accurately predicted infarct size at 24 hours after reperfusion., Conclusions: Early BBBD in focal cerebral ischemia initiates multifocally in the distal capillary and venular bed of the cerebral microvasculature. It is closely associated with perimicrovascular vasogenic edema and microglial activation and predicts the extent of final infarction., (© 2018 American Heart Association, Inc.)